1. Technical Field of the Invention
The present invention relates to modified DFR nucleic acids and encoding the modified DFR that preferentially utilize DHK as a substrate and their uses for genetically altering plants to increase the content of pelargonidin-based pigments in the plants.
2. Description of the Prior Art
Anthocyanins are classes of pigments that determine flower color and plant pigmentation in angiosperm plants. Among anthocyanins, pelargonidin-based pigments confer bric-red/orange color to plants, while cyanidin- and delphinidin-based pigments confer red and violet color each (Holton, et al. Plant Cell 7:1071-1083 (1995); Tanaka, et al. Plant Cell Physiol. 39:1119-1126 (1998)). Different ratio of these pigments confers a wide range of flower color. Many anthocyanin biosynthetic genes have been identified. One of key enzyme in the biosynthetic pathway is dihydroflavonol 4-reductase (DFR). The enzyme converts dihydroflavonols (dihydrokaempferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM)) to leucocyanidins. The leucocyanidins are subsequently converted to anthocyanins by other enzymes. The conversion of DHK to DHQ and DHM are catalyzed by flavonoid 3xe2x80x2-hydroxylase (F3xe2x80x2H) and flavonoid 3xe2x80x2,5xe2x80x2-hydroxylase (F3xe2x80x25xe2x80x2H). Since DFRs in most plants can convert all three dihydroflavonols to leucocyanidins, the ratio of three classes of anthocyanin pigments are mainly determined by the activity of F3xe2x80x2H and F3xe2x80x25xe2x80x2H (Holton, et al. Plant Cell 7:1071-1083 (1995)).
Since pelargonidin-based pigments confer the orange color to flowers, the F3xe2x80x2H and F3xe2x80x25xe2x80x2H activities must be absent for a plant to have orange colored flowers (U.S. Pat. No. 5,410,096). In many plant species, F3xe2x80x2H and F3xe2x80x25xe2x80x2H are encoded by a multiple genes, thus the mutant lines that lack F3xe2x80x2H and F3xe2x80x25xe2x80x2H are not easily found. This partially accounts for the rarity of orange-colored flowers in some plant species. Inability to reduce DHK to leucocyanidin by DFR in some species can also cause the lack of orange-colored flower. For example, DFRs from Petunia and Cymbidium convert DHK to its leucocyanidin very inefficiently, thus these species do not accumulate large ratio of pelargonidin-based anthocyanins even if F3xe2x80x2H and F3xe2x80x25xe2x80x2H are absent (Gerats, et al. Planta 155:364-368 (1982); Johnson, et al. Plant J. 19:81-85 (1999)). An orange-colored Petunia was engineered by introducing a maize DFR to a special mutant line of Petunia that lacks F3xe2x80x2H and F3xe2x80x25xe2x80x2H (Meyer, et al. Nature 330:677-678 (1987)). Since the maize DFR can convert all three dihydroflavonols to their leucocyanidins, such a mutant line that accumulates DHK was necessary for the development of orange-colored Petunia. The necessity of the special mutant line can be circumvented by using a DFR that utilizes DHK preferentially over DHQ and DHM.
Using chimeric DFRs between Petunia and Gerbera DFRs, we identified a region that determines the substrate specificity of DFR. By altering an amino acid in the region, we developed a DHK-specific DFR that converts DHK preferentially over DHQ and DHM. When expressed in plants, the DHK-specific DFR increases the pelargonidin-based pigments regardless of F3xe2x80x2H activity.
Accordingly, the object of this invention is to provide substrate-specific DFRs which have point mutations at residue number 134 of SEQ ID NO: 2 when the amino acids are aligned with the ClustalW program.
It is an also object herein to provide a DHK-specific DFR and nucleic acids encoding the DHK-specific DFR.
Still further, it is an object herein to provide transgenic plants expressing the DHK-specific DFR which confers a phenotype characterized by the increased content of pelargonidin-based pigments in the plants.
In accordance with the objects, the invention includes the modified DFRs and nucleic acids encoding the modified DFRs which have altered amino acid sequences at the substrate specificity determining region. The properties of modified DFRs are characterized by their abilities to reduce one substrate preferentially among DHK, DHQ, and DHM.
The invention also includes a modified DFR that reduces DHK preferentially over DHQ and DHM.
The invention also includes plants having at least one cell transformed with a vector comprising at least a portion of the modified DFR nucleic acids. Such plants have a phenotype characterized by the increased content of pelargonidin-based pigments.
The invention also includes vectors capable of transforming a plant cell to increase the content of pelargonidin-based pigments.
The invention also includes methods for producing plants having the increased content of pelargonidin-based pigments. The methods includes steps of transforming plant cells with vectors containing the modified DFR gene; regenerating plants from the transformed cells and selecting the plant having the increased content of pelargonidin-based-pigments.